1. Field of the Invention
This invention relates to methods for reversibly aggregating particles that are dispersed in a liquid medium by use of a polyionic polymer to aggregate the particles and a chemical reagent to reverse the aggregation of the particles by cleaving at least some of the bonds within the polyionic polymer. The invention has particular application to separation of cells from biological fluids such as whole blood, lymphatic fluid, urine, cell cultures, etc.
Numerous techniques are known for determining the presence and amount of an analyte in a sample, such as a biological fluid, for example, blood or urine. An in vitro assay procedure is the most common of these techniques. Many of these techniques involve competitive binding of the analyte to be determined and a labeled analog of such analyte to binding sites on a specific receptor, for example, an antibody. Some of these techniques involve an aggregation step where the bound or unbound labeled analog is bound to or associated with a support such as a particle, which becomes aggregated. The aggregate can then be examined for a signal produced in relation to the amount of analyte in the sample.
Several techniques are known for aggregating particles suspended in a liquid medium. For example, the particles may be aggregated by employing a polymer in the medium. In other instances, the particles may be co-aggregated with magnetic particles using a polymer which, for example, non-specifically binds the particles and the magnetic particles.
Several techniques are known for separating bound and unbound fractions. For example, such techniques include differential migration of the bound and the free fractions, e.g., chromatoelectrophereses, gel filtration, etc., chemical precipitation of the bound or free fraction, e.g., by means of organic solvents, salts, acids, etc. followed by filtration or centrifugation; immunological precipitation of the bound fraction, e.g. by double antibody technique followed by filtration or centrifugation; absorption of the bound or free fraction onto selective sorbing media, e.g., charcoal, silicates, resins, etc.; magnetic separation techniques, and the like.
Magnetic separations generally fall into two general categories. There are those separations in which the material to be separated is intrinsically magnetic. The second type involves rendering one or more components of a mixture magnetic by attachment to a magnetically responsive entity. In biological separations, for example, materials of interest are generally not sufficiently magnetic and therefore, magnetic particles bound to antibodies, lectins and other targeting molecules have been used to isolate many of these materials.
The binding of non-magnetic and magnetic particles to each other can be affected by pH. Therefore, one method that has been suggested for reversing the aggregation of the particles involves altering Binding can also be affected by other factors such as ionic strength and the presence of ionic or non-ionic polymers. In one approach, where the particles are bound by ionic interactions, the ionic strength is adjusted upwards to facilitate reversal of the coupling of the non-magnetic particles and the magnetic particles.
2. Description of the Related Art
A method for determining the concentration of substances in biological fluids (e.g., drugs, hormones, vitamins and enzymes) wherein magnetically responsive, permeable, solid, water insoluble, microparticles are employed is disclosed in U.S. Pat. No. 4,115,534. U.S. Pat. No. 4,452,773 discloses magnetic iron-dextran microspheres which can be covalently bonded to antibodies, enzymes and other biological molecules and used to label and separate cells and other biological particles and molecules by means of a magnetic field. Coated magnetizeable microparticles, reversible suspensions thereof, and processes relating thereto are disclosed in U.S. Pat. No. 4,454,234. A method of separating cationic from anionic beads in mixed resin beds employing a ferromagnetic material intrinsically incorporated with each of the ionic beads is described in U.S. Pat. No. 4,523,996. A magnetic separation method utilizing a colloid of magnetic particles is discussed in U.S. Pat. No. 4,526,681. UK Pat. Application GB 2,152,664A discloses magnetic assay reagents.
SUMMARY OF THE INVENTION
The method of the present invention is directed to the reversible aggregation of particles suspended in a liquid medium by employing a polyionic polymer to aggregate the particles and by contacting the aggregated particles with a chemical reagent capable of reversing the aggregation by cleaving the polyionic polymer. Where the particles in the medium are non-magnetic, they may form aggregates with each other, with other non-magnetic particles or with magnetic particles by addition of a polyionic polymer. Where the particles are magnetic, they may form aggregates with each other or with non-magnetic particles by addition of a polyionic polymer.
The method of the present invention has particular application in the assay of organic or biological analytes, particularly those analytes of interest in the analysis of body fluids. Of special interest are assays where the analyte is a member of a specific binding pair (sbp) where the analyte or an sbp member complementary to the analyte is bound, or can become bound, to the exterior surface of a particle. If the sbp member on the particle is not complementary to the analyte, then a complementary sbp member is also added. The method involves combining the particles which are suspended in a liquid medium with an sbp member complementary to the analyte or to the sbp member on the surface and adding a polyionic polymer that is capable of non-specifically aggregating the particles. After aggregation has occurred, a chemical reagent that is capable of reversing the aggregation by cleaving at least some of the bonds of the polyionic polymer is added. Thereafter, the residual specific aggregation of the particles is measured. Normally, the sbp member is detected by virtue of a signal created by the use of a signal producing system that generates a signal in relation to the amount of the analyte in the sample.
Of special interest are methods such as removing cells from whole blood, where the analyte is a surface component or becomes bound to a non-magnetic particle. In such an instance, the method involves combining in the medium the sample including non-magnetic particles, such as whole blood, magnetic particles and a polyionic polymer for non-specifically agglutinating the magnetic particles and the non-magnetic particles, e.g. the cells. The medium is subjected to a magnetic field gradient to separate the agglutinated cells from blood plasma. The agglutinated cells are contacted with a chemical reagent under conditions for reversing the agglutination by at least partial depolymerization of the polyionic polymer.
The method of the invention provides a way of separating non-magnetic particles from a medium by virtue of non-specifically aggregating such particles to magnetic particles by employing a polyionic polymer. It also provides for reversing the aggregation by employing a chemical reagent that cleaves at least some of the bonds within the polyionic polymer.
The present invention also includes novel polyionic polymers including polycations of the formula: EQU (A).sub.n
wherein A is positively charged and has 4 to 30 atoms other than hydrogen, wherein the atoms are independently selected from the group consisting of carbon, oxygen, phosphorous, nitrogen and sulfur and wherein at least one of the A groups has a cleavable bond; and n is on the average 5 to 10,000. PA1 wherein A is positively charged and has 4 to 30 atoms other than hydrogen, wherein the atoms are independently selected from the group consisting of carbon, oxygen, phosphorous, nitrogen and sulfur, and wherein at least one of said A groups has a cleavable bond; and n is an average of 5 to 10,000. PA1 wherein A is positively charged and has 4 to 30 atoms other than hydrogen, where the atoms are independently selected from the group consisting of carbon, oxygen, phosphorous, nitrogen and sulfur, and at least one of the A groups has a cleavable bond; and n is an average of 5 to 10,000. PA1 B is a linking group containing 2 to 30 atoms other than hydrogen which atoms are independently selected from the group consisting of carbon, oxygen, phosphorous, nitrogen and sulfur, and at least one of said B groups has a cleavable bond; and n is an average of 5 to 10,000. Preferably the cleavable bonds include disulfides or glycols.
Additionally, the invention includes kits for conducting the method of the invention and for conducting an assay for determining an analyte in a sample suspected of containing an analyte.